A novel keratan sulphate domain preferentially expressed on the large aggregating proteoglycan from human articular cartilage is recognized by the monoclonal antibody 3D12/H7.

Monoclonal antibodies (mAbs) were prepared against aggrecan which has been isolated from human articular cartilage and purified by several chromatographic steps. One of these mAbs, the aggrecan-specific mAb 3D12/H7, was selected for further characterization. The data presented indicate that this mAb recognizes a novel domain of keratan sulphate chains from aggrecan: (1) immunochemical staining of aggrecan is abolished by treatment with keratanase/keratanase II, but not with keratanase or chondroitin sulphate lyase AC/ABC; (2) after chemical deglycosylation of aggrecan no staining of the core-protein was observed; (3) different immunochemical reactivity was observed against keratan sulphates from articular cartilage, intervertebral disc and cornea for the mAbs 3D12/H7 and 5D4. For further characterization of the epitope, reduced and 3H-labelled keratan sulphate chains were prepared. In an IEF-gel-shift assay it was shown that the 3H-labelled oligosaccharides obtained after keratanase digestion of reduced and 3H-labelled keratan sulphate chains were recognized by the mAb 3D12/H7. Thus it can be concluded that the mAb 3D12/H7 recognizes an epitope in the linkage region present in, at least some, keratan sulphate chains of the large aggregating proteoglycan from human articular cartilage. Moreover, this domain seems to be expressed preferentially on those keratan sulphate chains which occur in the chondroitin sulphate-rich region of aggrecan, since the antibody does not recognize the keratan sulphate-rich region obtained after combined chondroitinase AC/ABC and trypsin digestion of aggrecan.

Development of enzyme immunoassays specific for keratan sulphate- and core-protein-epitopes of the large aggregating proteoglycan from human articular cartilage.

In the course of chronic inflammatory and degenerative joint diseases proteoglycans are degraded by the action of proteases and oxygen radicals. Therefore, proteoglycan fragments, released from cartilage into the peripheral blood, might be useful markers of cartilage degradation. Sensitive enzyme immunoassays are useful for the detection of these proteoglycan fragments in serum. We therefore developed specific monoclonal antibodies against the large aggregating proteoglycan (aggrecan), which has been isolated and purified from human articular cartilage. Two monoclonal antibodies which recognize a novel cartilage-specific epitope on the keratan sulphate chain of aggrecan (mAb 4B3/D10) and an epitope of the core-protein of aggrecan (4G4/A10) were selected for the development of competitive enzyme-immunoassays. These assays allow the sensitive and specific detection of cartilage-derived proteoglycan fragments, not only in synovial fluid but also in serum. They can now be used for the study of inflammatory and degenerative joint diseases.